Radioimmunoconjugates Targeting Phosphatidylserine for Use in the Treatment of Cancer

This application is a § 371 National Stage of International application no. PCT/US2022/075553 filed Aug. 28, 2022, which claims priority to U.S. provisional application Ser. No. 63/237,854 filed Aug. 27, 2021 which is hereby incorporated by reference in its entirety.

The instant application contains a Sequence Listing which has been submitted electronically in XML format in accordance with WIPO Standard ST.26 and is hereby incorporated by reference in its entirety. Said XML copy, created on Jul. 21, 2025, is named ATNM-004PCT_SL_ST26.xml and is 268,046 bytes in size.

The present disclosure relates to the field of targeted radiotherapeutics for the treatment or prevention of cancer.

Phosphatidylserine is a negatively charged glycerophospholipid that constitutes part of the bilayer plasma membrane of mammalian cells. Within the plasma membrane, phosphatidylserine is maintained predominantly on the inner leaflet, with the asymmetric distribution pattern being preserved by enzymes called flippases and floppases, which translocate phosphatidylserine from outer-to-inner leaflet and inner-to-outer leaflet, respectively. This asymmetry is essential because the negative charge on phosphatidylserine grants certain biological properties that help maintain normal cellular function: membrane curvature and charge-dependent docking of a large number of proteins essential for cytoskeletal reorganization and vesicular trafficking. However, when cells undergo apoptotic cell death, phosphatidylserine becomes increasingly externally facing through the action of scramblases, which redistribute phospholipids across the inner and outer leaflets, with the net effect of exposing more phosphatidylserine on the outer leaflet side of the cell membrane and, thus, on the cell surface. The externalized phosphatidylserine then serves as a marker for macrophage and dendritic cell engulfment, maintaining tissue homeostasis through removal of old or damaged cells without instigating an inflammatory response. This immunologically “silent” pathway helps prevent autoimmunity or otherwise deleterious inflammation, especially in tissues with high turnover rate. In fact, efferocytosis (phagocytosis of apoptotic cells) leads to immune tolerance, and cancer cells exploit this mechanism to evade antitumor immunity.

What is needed and provided by various aspects of the present invention disclosed herein are new and improved compositions and methods for treating proliferative disorders such as cancers and precancerous proliferative disorders.

The present disclosure provides uses of radiolabeled phosphatidylserine targeting agents in the diagnosis and treatment of proliferative disorders such as cancers and precancerous proliferative disorders. The radiolabeled phosphatidylserine targeting agents may, for example, include radiolabeled phosphatidylserine receptors, radiolabeled antibodies or antibody fragments that specifically bind phosphatidylserine or molecular complexes that include phosphatidylserine, radiolabeled small domain proteins such as a DARPin, anticalin, or affimer, or a radiolabeled peptides, aptamers, or small molecules that bind phosphatidylserine, and by binding phosphatidylserine externally presented by cancerous or precancerous cells can deliver DNA damage inducing radiation to said cells and neighboring cells.

The radiolabeled phosphatidylserine targeting agents useful for therapeutic interventions may, for example, include one or more radionuclides selected fromI,I,I,Y,Lu,Re,Re,Sr,Sm,P,Ac,Po,At,Bi,Bi,Ra,Th,Tb,Tb,Sc,Cu,Ce,Cs,Pb, andPd. In a related aspect, the radiolabeled phosphatidylserine targeting agents useful for therapeutic interventions may, for example, include a radionuclide which isI,Y,Lu,Ac,Bi,At,Th, orPb, or any combination thereof.

Therapeutic methods of the present disclosure include administering to a mammalian subject, such as a human patient, an effective amount of a radiolabeled phosphatidylserine targeting agent, alone or in combination with other cancer therapeutic agents and/or other cancer treatments. The effective amount may, for example, be a maximum tolerated dose (MTD), or a fractioned dose wherein the total amount of radiation administered in the fractioned doses is the MTD.

The radiolabeled phosphatidylserine targeting agent may, for example, be provided as a composition that includes a radiolabeled fraction and a non-radiolabeled fraction of the phosphatidylserine targeting agent. As such, for phosphatidylserine targeting agents that are proteins, such as antibodies and antibody fragments, an effective amount of the radiolabeled phosphatidylserine targeting agent may, for example, include a total protein dose of 1-100 mg or 1 to less than 100 mg, such as from 1 mg to 60 mg, or 5 mg to 45 mg. The total protein dose may, for example, be from 0.001 mg/kg to 3 mg/kg body weight of the subject, such as from 0.005 mg/kg to 2 mg/kg body weight of the subject. The total protein dose may, for example, be at or less than 2 mg/kg, or at or less than 1 mg/kg, or at or less than 0.5 mg/kg, or at or less than 0.1 mg/kg.

An effective amount of a radiolabeled phosphatidylserine targeting agent, such as anAc-anti-phosphatidylserine antibody, antibody fragment, binding protein, peptide, or small molecule, may, for example, include a radiation dose of 0.1 to 50 uCi/kg body weight of the subject, such as 0.1 to 5 uCi/kg body weight of the subject, or 5 to 20 uCi/kg subject body weight, or a radiation dose of 2 μCi to 2 mCi, or 2 μCi to 250 μCi, or 75 μCi to 400 μCi in a fixed (non-weight-based) radiation dose.

An effective amount of a radiolabeled phosphatidylserine targeting agent, such as anLu-anti-phosphatidylserine antibody, antibody fragment, binding protein, peptide, or small molecule, may, for example, include a radiation dose of 1 to 1000 μCi/kg body weight of the subject, such as 5 to 250 μCi/kg body weight of the subject, or 50 to 450 μCi/kg body weight, or a radiation dose of 10 mCi to 30 mCi, or 100 μCi to 3 mCi, or 3 mCi to 30 mCi in a fixed (non-weight-based) radiation dose.

An effective amount of a radiolabeled phosphatidylserine targeting agent, such as anI-labeled anti-phosphatidylserine antibody, antibody fragment, binding protein, peptide, or small molecule, may, for example, include a dose of at or below 1200 mCi in a fixed (non-weight-based) radiation dose, such as from at least 1 mCi to 1200 mCi, 1 mCi to at or below 100 mCi, or at least 10 mCi to at or below 200 mCi.

The effective amount of the radiolabeled phosphatidylserine targeting agent, may depend on the configuration of the targeting agent, i.e., full length protein or antibody, or antibody fragment (e.g., minibody, nanobody, etc.). For example, when the radiolabeled phosphatidylserine targeting agent includes anAc-labeled phosphatidylserine targeting agent that is a full-length antibody (such as mammalian IgG), the dose may, for example, be at or below 5 μCi/kg body weight of the subject, such as 0.1 to 5 μCi/kg body weight of the subject. Alternatively, when the phosphatidylserine targeting agent includes anAc-labeled phosphatidylserine targeting agent that is an antibody fragment, small domain protein such as a DARPin, anticalin, affimer, peptide, or aptamer, or small molecule, the dose may, for example, be greater than 5 μCi/kg body weight of the subject, such as 5 to 20 μCi/kg body weight of the subject, since such molecules are typically eliminated more quickly from the body than full-length antibodies.

The radiolabeled phosphatidylserine targeting agent may, for example, be administered according to a dosing schedule of one dose every 5, 7, 10, 12, 14, 20, 24, 28, 35, and 42 days throughout a treatment period, wherein the treatment period includes at least two doses.

The radiolabeled phosphatidylserine targeting agent may, for example, be administered according to a dose schedule that includes 2 doses, such as on days 1 and 5, 6, 7, 8, 9, or 10 of a treatment period, or days 1 and 8 of a treatment period.

The radiolabeled phosphatidylserine targeting agent may, for example, be administered as a single bolus or single infusion, such as an intravenous infusion.

Each administration of the radiolabeled phosphatidylserine targeting agent may, for example, be administered in a subject-specific dose, wherein each of a protein dose and a radiation dose are selected based on subject specific characteristics (e.g., weight, age, gender, health status, nature and severity of the cancer or tumor, etc.).

The methods may, for example, further include administration of one or more further cancer therapeutic agents, such as a chemotherapeutic agent, an anti-inflammatory agent, an immunosuppressive agent, an immunomodulatory agent, an antimyeloma agent, a cytokine, or any combination thereof. Exemplary chemotherapeutic agents that may be used include radiosensitizers that may synergize with the radiolabeled phosphatidylserine, such as temozolomide, cisplatin, and/or fluorouracil.

The methods may, for example, further include administration of one or more immune checkpoint therapies. Exemplary immune checkpoint therapies include an monoclonal antibody or other blocking agent against CTLA-4, PD-1, TIM3, VISTA, BTLA, LAG-3, TIGIT, CD28, OX40, GITR, CD137, CD40, CD40L, CD27, HVEM, PD-L1, PD-L2, PD-L3, PD-L4, CD80, CD86, CD137-L, GITR-L, CD226, B7-H3, B7-H4, BTLA, TIGIT, GALS, KIR, 2B4, CD160, A2aR, CGEN-15049, or any combination thereof. The immune checkpoint therapy may, for example, include an antibody or other blocking agent against an immune checkpoint protein selected from the group consisting of an antibody against PD-1, PD-L1, CTLA-4, TIM3, LAG3, VISTA, and any combination thereof. The immune checkpoint therapy may, for example, be provided in a subject effective amount including a dose of 0.1 mg/kg to 50 mg/kg of the patient's body weight, such as 0.1-5 mg/kg, or 5-30 mg/kg.

The methods may for example, further include administration of one or more CD47 blockades. The CD47 blockade may, for example, include a monoclonal antibody or other blocking agent that prevents CD47 binding to SIRPα, such as magrolimab, lemzoparlimab, AO-176, AK117, IMC-002, IBI-188, IBI-322, BI 766063, ZL-1201, AXL148, ES004, SRF231, SHR-1603, TJC4, TTI-621, or TTI-622. Exemplary effective doses for the CD47 blockade include 0.05 to 5 mg/kg patient weight. The CD47 blockade may, for example, include agents that modulate the expression of CD47 and/or SIRPα, for example, by an antisense nucleic acid approach. An exemplary agent includes phosphorodiamidate morpholino oligomers (PMO) that block translation of CD47, such as MBT-001. The CD47 blockade may, for example, include a small molecule inhibitor such as RRx-001.

The methods may, for example, further include administration of one or more DNA damage response inhibitors (DDRi). An exemplary DDRi includes at least one or more antibodies or small molecules targeting poly(ADP-ribose) polymerase (i.e., a poly(ADP-ribose) polymerase inhibitor or PARPi). The PARPi may, for example, be a small molecule therapeutic selected from the group consisting of olaparib, niraparib, rucaparib, talazoparib, or any combination thereof. The PARPi may, for example, be provided in a subject effective amount including 0.1 mg/day-1200 mg/day, such as 0.100 mg/day-600 mg/day, or 0.25 mg/day-1 mg/day. Exemplary subject effective amounts include 0.1 mg, 0.25 mg, 0.5 mg, 0.75 mg, 1.0 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 750 mg, 800 mg, 900 mg, and 1000 mg, taken orally in one or two doses per day. Another exemplary DDRi includes an inhibitor of Ataxia telangiectasia mutated (ATM), Ataxia talangiectasia mutated and Rad-3 related (ATR), or Wee1. Exemplary inhibitors of ATM include KU-55933, KU-59403, wortmannin, CP466722, and KU-60019. Exemplary inhibitors of ATR include at least Schisandrin B, NU6027, NVP-BEA235, VE-821, VE-822, AZ20, and AZD6738. Exemplary inhibitors of Wee1 include AZD-1775 (i.e., adavosertib).

The methods may, for example, further include administration of a radiation cancer treatment such as external beam radiation and/or brachytherapy.

The methods may, for example, further include administration of any combination of the further therapeutic agents or modalities set forth herein. Exemplary combinations include any combination of at least one or more DDRi, one or more immune checkpoint therapies, one or more CD47 blockades, one or more chemotherapeutics, one or more therapeutic targeting agents (e.g. therapeutic antibodies, antibody drug conjugates, or radiolabeled targeting agents against targets other than phosphatidylserine), and one or more radiation therapies (e.g., external beam radiation or brachytherapy).

The radiolabeled phosphatidylserine targeting agent and the one or more further therapeutic agents and/or treatments may be administered simultaneously or sequentially or in an overlapping manner. It should be understood that when more than one therapeutic agent are administered to a subject sequentially, there may nevertheless be a period of overlapping activity and/or resulting effects of the agents within the subject.

The phosphatidylserine targeting agent may, for example, include a multi-specific targeting agent, such as a multi-specific antibody, in which a portion/part of the agent recognizes or otherwise targets phosphatidylserine. Thus, the methods may include administering to the subject an effective amount of a radiolabeled multi-specific targeting agent (such as antibody), wherein the multi-specific targeting agent (such as antibody) includes: a first target recognition component that specifically binds to cell surface phosphatidylserine (or a complex including it), and a second target recognition component that binds to a different epitope of the phosphatidylserine (or complex including it) as the first target recognition component and/or to one or more further (non-phosphatidylserine) antigens, such as one or more cancer cell-associated antigens or other cancer-associated antigens. A radiolabeled phosphatidylserine targeting agent may, for example, include or be a multi-specific targeting agent, such as antibody, having specific binding activity against phosphatidylserine (or a complex including it) and against one or more further antigens, such as one or more cancer cell-associated antigens or other cancer-associated antigens. In the case of a radiolabeled multi-specific targeting agent, any part or portion of the targeting agent may be radiolabeled.

Additional features, advantages, and aspects of the invention may be set forth or apparent from consideration of the following detailed description and claims. Moreover, it is to be understood that both the foregoing summary of the invention and the following detailed description are exemplary and intended to provide further explanation without limiting the scope of the invention as claimed.

The present disclosure provides compositions and methods for treating cancers and precancerous proliferative disorders by administering to a subject in need of treatment therefor a radiolabeled phosphatidylserine targeting agent in order to deliver lethal radiation to cancerous and/or precancerous cells having phosphatidylserine exposed on their cell surface (cell surface phosphatidylserine). A related aspect of the invention includes radiolabeling a phosphatidylserine-targeting agent to produce a radiolabeled phosphatidylserine targeting agent for use in delivering lethal radiation to cancer cells or precancerous cells that express cell surface phosphatidylserine. Various types of phosphatidylserine binding agents such as monoclonal antibodies, antigen-binding antibody fragments, binding proteins, antibody mimetics, other proteins, peptides, or small molecules, can be labeled with radionuclides for use in causing DNA damage and subsequent cell death of target cells expressing cell surface phosphatidylserine.

By conjugating a radioactive payload to the phosphatidylserine-targeting agent, such as via a stable metal chelator such as DOTA, radiation can be delivered specifically and systemically to primary tumors, metastatic tumors and cancer cells or precancerous cells generally, which often remain undetected and are not amenable to treatment by external beam radiation, while minimizing exposure of healthy tissues that do not significantly express cell surface phosphatidylserine.

Radio-conjugation/radiolabeling of targeting agents such as antibodies has multiple advantages over drug conjugation. Unlike drug conjugates, radio-conjugates do not require internalization because the emitted radiation can penetrate cells. For example, alpha particles can cross multiple cellular membranes to reach the cell nuclei, causing clusters of dsDNA breaks that are not easily repaired (Nelson, 2020). Furthermore, whereas antibody-drug conjugates require high surface density of the targeted molecule to deliver sufficient quantities of the toxic payload (Sadekar, 2015), radioligands are less sensitive to target expression level since, for example, a single alpha particle is capable of inducing cancer cell death (Neti, 2006). “Cross-firing” is another advantage of radio-conjugates, whereby radiation is delivered to both the targeted cancer cells and adjacent malignant cells (Haberkorn, 2017). In this manner, radioimmunotherapy can exert clinical efficacy even if the target expression profile is heterogeneous within the tumor.

Lastly, targeting phosphatidylserine using radioimmunotherapy is advantageous for another important reason. Radiation delivered by the radiolabeled phosphatidylserine targeting agent itself increases the cell surface expression of phosphatidylserine, leading to a feed-forward mechanism that drives further accumulation of the radiolabeled phosphatidylserine targeting agent at target lesions to enhance its therapeutic effect. And, since cell surface expression of phosphatidylserine is upregulated in response to cell damage and stress, radiolabeled phosphatidylserine targeting agents may also be used in combination with other anticancer therapies to amplify overall efficacy in a synergistic manner.

In this regard, therapeutically useful radionuclides include, but are not limited to, Actinium-225, Astatine-211, Bismuth-213, Iodine-131, Lead-212, Lutetium-177, Radium-223, Thorium-227, Yttrium-90. Of these, Actinium-225 (Ac) displays characteristics that render it particularly well suited for anticancer therapy.

Ac emits four high linear energy transfer alpha particles during its decay profile over a very short distance of about 3-4 cells' thickness (Pouget, 2011), making this payload very potent in causing lethal double-strand DNA (dsDNA) breaks by direct ionizing radiation. This short path length also makesAc safer to handle compared to beta-emitting isotopes that have longer ranges (Nelson, 2020). Labeling an antibody withAc substantially decreases the amount of total antibody necessary to achieve a tumor response. Based on previous experience comparing the efficacy ofAc-labeled and unlabeled therapeutic monoclonal antibodies (Dawicki, 2019), the amount of antibody required to elicit a tumor response may be decreased approximately 30-fold forAc-labeled antibody versus unlabeled therapeutic antibody. Furthermore, given the potency of the alpha-emitter, a single administration of radiolabeled targeting agent can be sufficient to observe tumor reduction.

Accordingly, the present disclosure provides novel compositions and methods for treating proliferative disorders, such as cancers and precancerous proliferative disorders, using radiolabeled phosphatidylserine targeting agents to target cancerous and/or precancerous cells expressing, such as overexpressing versus normal cells, cell surface phosphatidylserine. The methods generally include administering to a mammalian subject, such as a human patient, in need of treatment for a cancer or precancerous proliferative disorder an effective amount of a radiolabeled phosphatidylserine targeting agent, such as a radiolabeled antibody, antibody fragment, binding protein antibody mimetic, peptide, or small molecule that specifically binds to phosphatidylserine (or to a complex including phosphatidylserine), alone or in combination or conjunction with one or more additional therapeutic agents or treatments.

The additional therapeutic agents or treatments may, for example, include one or more of: one or more immune checkpoint therapies, one or more inhibitors of a component of the DNA damage response pathway (i.e., a DNA damage response inhibitor, DDRi, such as one or more agents against poly(ADP-ribose) polymerase, i.e., PARPi), one or more CD47/SIRPα axis blockades, one or more chemotherapeutic agents such as radiosensitizers or cytotoxic agents, one or more enzyme inhibitors such as kinase inhibitors, one or more anti-inflammatory agents, one or more an immunosuppressive agents, one or more immunomodulatory agents, one or more antimyeloma agents, one or more cytokines, one or more therapeutic targeting agents (e.g. therapeutic antibodies, antibody drug conjugates, or radiolabeled targeting agents against targets other than phosphatidylserine), and one or more radiation therapies (e.g., external beam radiation or brachytherapy).

The singular forms “a,” “an,” “the” and the like include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an” antibody includes both a single antibody and a plurality of different antibodies.

The words “comprising” and forms of the word “comprising” as well as the word “including” and forms of the word “including,” as used in this description and in the claims, do not limit the inclusion of elements beyond what is referred to. Additionally, although throughout the present disclosure various aspects or elements thereof are described in terms of “including” or “comprising,” corresponding aspects or elements thereof described in terms of “consisting essentially of” or “consisting of” are similarly disclosed. For example, while certain aspects of the invention have been described in terms of a method “including” or “comprising” administering a radiolabeled targeting agent, corresponding methods instead reciting “consisting essentially of” or “consisting of” administering the radiolabeled target are also within the scope of said aspects and disclosed by this disclosure.

The term “about” when used in this disclosure in connection with a numerical designation or value, e.g., in describing temperature, time, amount, and concentration, including in the description of a range, indicates a variance of ±10% and, within that larger variance, variances of ±5% or ±1% of the numerical designation or value.

As used herein, “administer”, with respect to a targeting agent (such as an antibody, antibody fragment, binding protein, Fab fragment, peptide, or aptamer) or other therapeutic agents means to deliver the agent to a subject's body via any known method suitable for the agent. Specific modes of administration include, without limitation, intravenous, transdermal, subcutaneous, intraperitoneal, intrathecal and intra-tumoral administration. Exemplary administration methods for antibodies may be as substantially described in International Publication No. WO 2016/187514, incorporated by reference herein.

In addition, in this disclosure, targeting agents such as antibodies may be formulated using one or more routinely used pharmaceutically acceptable carriers or excipients. Such carriers are well known to those skilled in the art. For example, injectable drug delivery systems include solutions, suspensions, gels, microspheres and polymeric injectables, and can include excipients such as solubility-altering agents (e.g., ethanol, propylene glycol and sucrose) and polymers (e.g., polycaprylactones and PLGA's).

As used herein, the term “antibody” includes, without limitation, (a) an immunoglobulin molecule including two heavy chains and two light chains and which recognizes an antigen; (b) polyclonal and monoclonal immunoglobulin molecules; (c) monovalent and divalent fragments thereof, such as Fab, di-Fab, scFvs, diabodies, minibodies, and single domain antibodies (sdAb) such as nanobodies; (d) naturally occurring and non-naturally occurring, such as wholly synthetic antibodies, IgG-Fc-silent, and chimeric antibodies; and (e) bi-specific forms thereof. Immunoglobulin molecules may derive from any of the commonly known classes, including but not limited to IgA, secretory IgA, IgG and IgM. IgG subclasses are also well known to those in the art and include, but are not limited to, human IgG1, IgG2, IgG3 and IgG4. The N-terminus of each chain defines a “variable region” of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these regions of light and heavy chains respectively. Antibodies used may, for example, be human, humanized, nonhuman or chimeric. When a specific aspect of the present disclosure refers to or recites an “antibody,” it is envisioned as referring to any of the full-length antibodies or fragments thereof disclosed herein, unless explicitly denoted otherwise.

A “humanized” antibody refers to an antibody in which some, most or all amino acids outside the CDR domains of a non-human antibody are replaced with corresponding amino acids derived from human immunoglobulins. In one embodiment of a humanized form of an antibody, some, most or all of the amino acids outside the CDR domains have been replaced with amino acids typical of human immunoglobulins, whereas some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they do not abrogate the ability of the antibody to bind to a particular antigen. A “humanized” antibody retains an antigenic specificity similar to that of the original antibody.

A “chimeric antibody” refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species, such as an antibody in which the variable regions are derived from a mouse antibody and the constant regions are derived from a human antibody. For example, one type of chimeric antibody that may be used as a targeting agent in the various aspects of the invention is an immunoglobulin such as IgG consisting of non-human, such as mouse or rat, variable domains/regions (such as VH and VL) and a human Fc domain.

A “complementarity-determining region”, or “CDR”, refers to amino acid sequences that, together, define the binding affinity and specificity of the variable region of an immunoglobulin antigen-binding site. There are three CDRs in each of the light and heavy chains of an antibody. The CDRs (and framework regions) in the amino acid sequence of an antibody may, for example, be delineated according to the Kabat or IMGT numbering conventions.

A “framework region” or “FR”, refers to amino acid sequences surrounding and interposed between CDRs, typically conserved, that act as the “scaffold” for the CDRs.

A “constant region” refers to the portion of an antibody molecule that is consistent for a class of antibodies and is defined by the type of light and heavy chains. For example, a light chain constant region can be of the kappa or lambda chain type and a heavy chain constant region can be of one of the five chain isotypes: alpha, delta, epsilon, gamma or mu. This constant region, in general, can confer effector functions exhibited by the antibodies. Heavy chains of various subclasses (such as the IgG subclass of heavy chains) are mainly responsible for different effector functions.

As used herein, a “phosphatidylserine targeting agent” may, for example, be an antibody as defined herein, e.g., full-length antibody such as a monoclonal IgG antibody, antibody fragment, minibody, nanobody, etc., that binds to phosphatidylserine or to a complex that includes phosphatidylserine (such as a complex of phosphatidylserine and β2GP1 protein) with a high immunoreactivity. A phosphatidylserine targeting agent may, for example, be a phosphatidylserine binding protein or fusion protein that does not include the antigen recognition component(s) of an antibody and/or is not an antibody mimetic. A phosphatidylserine targeting agent may, for example, be or include a small domain protein such as a DARPin, anticalin, or affimer, or a peptide, aptamer, or small molecule that specifically binds to phosphatidylserine.

A “DARPin” is an antibody mimetic protein having high selectivity and high affinity for a specific protein. DARPins have a molecular weight of 14 to 21 kDa, consist of 2 to 5 ankyrin repeat motifs. They include a core region having a conserved amino acid sequence that provides structure and a variable target binding region that resides outside of the core and binds to a target. DARPins may further include an immune cell modulation motif, such as any described hereinabove.

An “Anticalin” is a scaffold protein that is a single-chain-based antibody mimetic capable of specifically binding to an antigen and typically having a size of about 20 kDa. Anticalin molecules are generated by combinatorial design from natural lipocalins, which are abundant plasma proteins in humans, and reveal a simple, compact fold dominated by a central 3-barrel, supporting four structurally variable loops that form a binding site.

An “Affimer” is a small, highly stable protein engineered to display peptide loops which provide a high affinity binding surface for a specific target protein. Affimer are derived from the cysteine protease inhibitor family of cystatins and typically have a low molecular weight of 12-14 kDa. Affimers are composed of a stable protein scaffold based on the cystatin protein fold. They display two peptide loops and an N-terminal sequence that can be randomized to bind different target proteins with high affinity and specificity similar to antibodies. Stabilization of the peptide upon the protein scaffold constrains the possible conformations which the peptide may take, thus increasing the binding affinity and specificity compared to libraries of free (non-constrained) peptides.

As used herein, an “Aptamer” is an at least partially single stranded polynucleic acid molecule that by virtue of its sequence composition can bind specifically to biosurfaces, a target compound or a moiety. Aptamers are highly specific, relatively small in size, and non-immunogenic. Aptamers may, for example, be selected using the biopanning method known as SELEX (Systematic Evolution of Ligands by Exponential enrichment). The SELEX process is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules and is described in, e.g., U.S. Pat. No. 5,270,163 (see also WO 91/19813) entitled “Nucleic Acid Ligands.” Each SELEX-identified nucleic acid ligand is a specific ligand of a given target compound or molecule. Methods of generating an aptamer for any given target are well known in the art.